Heart valve disease results in over 23,000 annual deaths in the United States, with calcific aortic valve disease (CAVD) being the most prevalent. There is no pharmacological treatment to prevent or reverse CAVD, due to the poor understanding of underlying causes and therefore surgical intervention remains the only effective option. Valve calcification is mediated by valve interstitial cells (VICs) which in response to pathological stimuli abnormally express bone development genes and differentiate into osteoblast-like cells termed osteoVICs. These cells then deposit a calcified matrix that limit cusp movement and leads to stenosis. Despite this, the mechanisms that promote differentiation of osteoVICs are unknown, but our data suggests that Sox9 plays a role. Our data shows that in non-diseased valves Sox9 is highly expressed in the nuclei of VICs where it prevents calcification by functioning as a transcription factor to repress osteogenic gene expression while activating chondrogenic mRNAs that make up healthy valve leaflets. In contrast, we observe reduced Sox9 nuclear localization in several models of calcification including excised valves from human patients and mouse models of CAVD, and in vitro calcification assays, suggesting that Sox9 nuclear export is a common feature of CAVD. Furthermore, our published in vivo work shows that reduced Sox9 function in mice promotes differentiation of osteoVICs and CAVD while attenuating the contribution of chondrogenic matrix proteins indicating a causative role for reduced Sox9 function in CAVD. While our published and preliminary data have identified Sox9 as a key regulator of CAVD, the mechanism underlying its nuclear localization in health and disease are not known, yet identifying this will provide insights for the development of mechanistic-based therapies beyond surgical intervention. In this proposal we demonstrate that in healthy valves, Tgf1 secreted from valve endothelial cells (VECs) molecularly communicates with Sox9 in VICs to retain nuclear localization and prevent calcification via RhoA kinase (ROCK) signaling. Therefore, we hypothesize that: In calcific aortic valve disease, abrogated expression of Tgf1 signaling molecules in VECs prevents ROCK-mediated phosphorylation and nuclear localization of Sox9 in VICs, leading to differentiation of osteoVICs and CAVD. To do this, we will use innovative in vitro, in vitro, genomic and clinical approaches to address the following three specific aims: 1) Determine the mechanism by which Tgf1 mediates Sox9 nuclear localization in healthy and osteogenic valve interstitial cells; 2) VECs isolated from mouse models of CAVD commonly demonstrate altered expression of Tgf signaling molecules associated with dysfunction; and 3) Determine if pharmacological and genetic targeting to prevent Sox9 nuclear export prevents CAVD in vivo. Findings from this proposal will for the first time define a molecular pathway that underlies CAVD and identify mechanistic- based therapies that can be developed to treat affected human patients.